American Society of Hematology

Fig. 5.

$Fig. 5. Two-dimensional phosphoamino acid analysis of p85 and p110 phosphorylated, in vitro. PI3K was immunoprecipitated from, post-130,000g cytosolic fractions of platelet lysates and the immune complexes subjected to an in vitro protein kinase assay in the presence of Mn2+ and γ-32P-ATP in the absence or presence of either 30 μmol/L LY294002 (A), or 5 μmol/L erbstatin-A (B). Radiolabeled proteins were eluted and separated by SDS-PAGE. p85 and p110 bands were subjected to two-dimensional phosphoamino acid analysis as described in Materials and Methods. Phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards were run concurrently with the p85 and p110 amino acid hydrolysates and stained with ninhydrin. Migration of phosphoamino acid standards was coincident with the radiolabeled spots.$

Two-dimensional phosphoamino acid analysis of p85 and p110 phosphorylated, in vitro. PI3K was immunoprecipitated from, post-130,000g cytosolic fractions of platelet lysates and the immune complexes subjected to an in vitro protein kinase assay in the presence of Mn²⁺ and γ-³²P-ATP in the absence or presence of either 30 μmol/L LY294002 (A), or 5 μmol/L erbstatin-A (B). Radiolabeled proteins were eluted and separated by SDS-PAGE. p85 and p110 bands were subjected to two-dimensional phosphoamino acid analysis as described in Materials and Methods. Phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards were run concurrently with the p85 and p110 amino acid hydrolysates and stained with ninhydrin. Migration of phosphoamino acid standards was coincident with the radiolabeled spots.

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